Valproic acid (VPA) has been reported to cause elevation of plasma phenobarbital (PB) levels in epileptic patients. We have found that VPA inhibits the metabolism of PB in epileptic patients and therefore may be responsible for this drug-drug interaction (Pharmacologist 21:208, 1979). We have further studied this interaction in an in vitro system, using hepatic microsomes from adult male PB-treated Holtzman rats. An assay system for microsomal parahydroxylation of PB was developed. Microsomes were incubated at 37 degrees C in the presence of 0.1 M potassium phosphate buffer (pH 7.50) and 0.25 mM NADPH. The microsomal reaction mixture was washed twice with benzene to remove substrate PB, and the p-hydroxyphenobarbital (p-OHPB) was then extracted with ethylacetate. The extract was perethylated with ethyl iodide in acetonitrile in the presence of tetraethylammonium hydroxide. The p-OHPB was quantitated by gas chromatography, using a nitrogen selective flame ionization detector, with diphenylbarbituric acid as the internal standard. VPA was shown to be a competitive inhibitor of microsomal parahydroxylation of PB. The Michaelis constant (Km) was 1.16 mM; maximal velocity (Vmax), 0.325 nmole p-OHPB formed min-l nmole P450-l; and inhibition constant (Ki), 1.20 mM.